Conjugated linoleic acid isomerase and a process for the production of conjugated linoleic acid

ABSTRACT

The present invention relates to a process for the production of conjugated linoleic acid and a process for the production of triglycerides with an increased content of conjugated linoleic acid. Moreover, the invention relates to a nucleic acid sequence; a nucleic acid construct, a vector and transgenic organisms comprising at least one nucleic acid sequence or one nucleic acid construct which encodes a polypeptide with conjugated linoleic isomerase activity. Furthermore, the invention relates to the use of a microorganism of the genus  Bifidobacterium  as a probiotic.

RELATED APPLICATIONS

This application claims the benefit of European Application Serial No.01113962.3, filed Jun. 8, 2001, the entire contents of which areincorporated herein by this reference.

FIELD OF THE INVENTION

The present invention relates to a process for the production ofconjugated linoleic acid and a process for the production oftriglycerides with an increased content of conjugated linoleic acid.

Moreover, the invention relates to a nucleic acid sequence; a nucleicacid construct, a vector and transgenic organisms comprising at leastone nucleic acid sequence or one nucleic acid construct which encodes apolypeptide with conjugated linoleic isomerase activity. Furthermore,the invention relates to the use of a microorganism of the genusBifidobacterium as a probiotic.

BACKGROUND

Fatty acids and triglycerides have a multiplicity of applications in thefood industry, animal nutrition, cosmetics and in the pharmaceuticalsector. Depending on whether they are free saturated or unsaturatedfatty acids or triglycerides with an increased content of saturated orunsaturated fatty acids, they are suitable for a very wide range ofapplications; thus, for example, polyunsaturated fatty acids are addedto baby formula to increase the nutritional value. The various fattyacids and triglycerides are obtained mainly from microorganisms such asMortierella or from oil-producing plants such as soya, oilseed rape,sunflowers and others, where they are usually obtained in the form oftheir triacyl glycerides. Alternatively, they are obtainedadvantageously from animals, such as fish. The free fatty acids areprepared advantageously by hydrolysis.

Whether oils with unsaturated or with saturated fatty acids arepreferred depends on the intended purpose; thus, for example, lipidswith unsaturated fatty acids, specifically polyunsaturated fatty acids,are preferred in human nutrition since they have a positive effect onthe cholesterol level in the blood and thus on the possibility of heartdisease. They are used in a variety of dietetic foodstuffs ormedicaments.

Especially valuable and sought-after unsaturated fatty acids are theso-called conjugated unsaturated fatty acids, such as conjugatedlinoleic acid. A series of positive effects have been found forconjugated fatty acids; thus, the administration of conjugated linoleicacid reduces body fat in humans and animals, and increases theconversion of feed into body weight in the case of animals (WO 94/16690,WO 96/06605, WO 97/46230, WO 97/46118). By administering conjugatedlinoleic acid, it is also possible to positively affect, for example,allergies (WO 97/32008) or cancer (Banni et al., Carcinogenesis, Vol.20, 1999: 1019–1024, Thompson et al., Cancer, Res., Vol. 57, 1997:5067–5072).

Conjugated linoleic acid (=CLA) is an intermediate of linoleic acidmetabolism in ruminants. CLA refers to a mixture of positional andgeometric isomers of linoleic acid, involving double bonds at positions9 and 11, 10 and 12 or 11 and 13, and has gained considerable attentionin recent years because of the many beneficial effects attributed to thecis-9, trans-11 and trans-10, cis-12 isomers, in particular. Theseinclude anti-carcinogenic activity, antiatherogenic activity, theability to reduce the catabolic effects of immune stimulation, theability to enhance growth promotion and the ability to reduce body fat(Martin and Banni, 1998 for review, and references therein). The isomerscan differ positionally (mainly at positions 7 and 9, 9 and 11; or 10and 12) (Ha et al., 1987) and geometrically (cis-cis, cis-trans,trans-cis, trans-trans). Of the individual isomers of CLA, cis-9,trans-11-octadecadienoic acid has been implicated as the mostbiologically active because it is the predominant isomer incorporatedinto the phospholipids of cell membranes, liver phospholipids andtriglycerides (Kramer et al., 1998). This is the only isomerincorporated into the phospholipid fraction of cell membranes of animalsfed a mixture of CLA isomers (Ha et al., 1990; Ip et al., 1991). Thisisomer is also the predominant dietary form of CLA, obtained from fatsderived from ruminant animals, including milk, dairy products and meat(Chin et al., 1992, O'Shea et al., 2000).

Studies have shown that CLA may have potential in the prevention of awide range of human medical conditions, and a number of potential healthbenefits have been described for CLA, including as mentioned aboveanticarcinogenic activity, antiatherogenic activity, potential in theprevention of diabetes, obesity and bone disorders. Given that dietaryCLA has the potential to beneficially affect human health, it isimportant to identify effective strategies to enrich the natural form ofCLA in food products. Currently, the effective level of dietary CLA fordisease prevention in humans is not known. Furthermore, the long-termhealth implications of a low dietary CLA intake at critical stagesthroughout life are unknown, as for example in formula-fed infants,compared with breast-fed infants, the latter group receiving arelatively higher CLA intake. In the rodent model, dietary CLA was moreeffective as an anticarcinogen when consumed during periods of activemammary gland development (Ip et al., Nutr. Cancer, 1995, 24: 241–247),which may indicate that increased CLA intake during adolescence mightpreferentially decrease the risk of cancer in women.

Few data concerning the CLA intake are available. In Germany the dailyCLA intake has been estimated to be 0.36 g/day for women and 0.44 g/dayfor men (Fritsche et al., 1998). CLA in human tissue is predominantlythe isomere cis-9, trans-11-octa-decadienoic acid (>95%), three minorisomers have also been identified. Trans-9, trans-11–18:2, cis-9,cis-11–18:2 and trans9, cis-11–18:2, [Fritsche et al., ZeitschriftLebensmittel. Untersuchung Forschung A-Food Research & Technology205:415–418 (1997)]. The origin is thought to be dietary and theconsumption of cheddar cheese, a good source of CLA, has been shown toenhance plasma CLA levels in men, [Huang et al. Nutr. Res. 14:373–386(1994)]. In another study it was found that the relationship betweenmilk fat intake and the occurence of cis-9, trans-11-octadecadienoicacid in human tissue was significantly correlated, [Jiang et al., Am. J.Clin. Nutr. 70:21–29 (1999)]. Safflower oil, a rich source of linoleicacid, did not increase plasma CLA levels suggesting that the intestinalflora of humans do not possess the ability to convert linoleic acid toconjugated linoleic acid, however a CLA increase was observed in somesubjects [Herbel et al., Am. J. Clin. Nutr. 67: 332–337 (1998)]. Dietarytrans fatty acid has been shown to increase serum CLA, [Salminen et al.,Nutritional Biochemistry 9: 93–98 (1998)]. It was in this studyconcluded that CLA may be formed by desaturation of trans fatty acidpossibly by a liver enzyme as has been described for rats.

The principial dietary sources of CLA are milk, dairy products and meatfrom ruminants, but as a result of differences in environmentalconditions and diet of the ruminant species, the CLA content of milk andbeef fat vary substantially [Michelle et al., Advances in ConjugatedLinoleic Acid Research, Volume 1 (1999)]. Among the richest dietarysources of CLA are milk, dairy products, beef and lamb (Chin et al., J.Food Comp. and Anal., 1992, 5: 185–197; Fritsche and Steinhart, Z.Lebensm. Unters. Forsch., 1998, A206: 77–82 and Lipid, 1998, 6S:190–210).

In fat from ruminant meats and dairy products, the cis-9, trans-11 CLAisomer is present at 80–90% of the total CLA isomers (Chin et al., J.Food Comp. and Anal., 1992, 5: 185–197).

As mentioned above the origin of CLA in foods is mainly due to thebiohydrogenation of dietary linoleic acid by anaerobic rumen bacteria.Accordingly the main dietary sources of CLA are meat from ruminantanimals and dairy products, and the main CLA isomer found is cis-9,trans-11-C18:2, (80–90%). In uncooked meats, lamb and beef answer forthe highest CLA levels 5.6 mg/g of fat in lamb and 4.3 mg/g of fat inbeef (Chin et al., 1992; Fritsche and Steinhart, 1998 ). CLA levels inmilk varies with season, highest values occuring when pastures are lushand rich in PUFAs, hence levels of CLA in dairy products such as cheesealso varies. In vegetable oils CLA is present in low amounts (0.2–0.7mg/g fat) and contain higher levels of the isomer trans-10, cis-12-C18:2(˜40%) (Chin et al., 1992). Since fatty acids with conjugated doublebonds are a well-known phenomena in plants, specific enzyme systems arebelived to be involved. Meats from non-ruminant animals can contain CLAin lower amounts and it may occur from dietary sources such as feedingmeat meal. It could also be explained by formation of CLA by intestinalflora as has been shown for rats (Chin et al., J. Nutr., 1993, 124:694–701). CLA can also be produced by free radical-based double bondshifting during autooxidation and during partial hydrogenation performedindustrially.

CLA can be manufactured synthetically from alkaline isomerization oflinoleic and linolenic acids, or vegetable oils containing linoleic orlinolenic acids. Two reactions are catalyzed when heating oil at 180° C.under alkaline conditions; hydrolysis of the fatty acid ester bond fromthe triglyceride lipid backbone, which produces free fatty acids, andconjugation of unconjugated unsaturated fatty acids with two or moreapproproiate double bonds (WO 99/32604). This method produces about20–35% cis-9, trans-11 CLA and about the same amount of trans-10, cis-12CLA, but enrichment of either of the isomers relative to the other ispossible by using a fractional crystallization procedure.

In addition other isomers are produced mainly trans, trans isomers.

The chemical preparation of conjugated fatty acids, for exampleconjugated linoleic acid, is also described in U.S. Pat. Nos. 3,356,699and 4,164,505.

The presence of conjugated unsaturated fatty acids in milk fat was firstestablished by Booth et al. Bioch. J. 29, 133–137 (1935), who alsoshowed that these fatty acids increased in milk fat when cows wereturned out to pasture after winter, by demonstrating an increasedabsorption of the fatty acids in the ultra-violet (UV) region at 230 nm.CLA is formed in the rumen during microbial biohydrogenation of dietarylinoleic acid (Kepler and Tove, 1969 Methods in enzymology Vol XIV,p105; J. Biol. Chem., Vol 246, No. 14, 1970: 3612–3620 and J. Biol.Chem., Vol 246, No. 9, 2765–2771). The complete biohydrogenation oflinoleic acid in the rumen is a three step process, yielding C 18:0,stearic acid, as an end product. The first reaction, the conversion oflinoleic acid to cis-9, trans-11 CLA by linoleic acid isomerase of rumenbacteria occurs very rapidly, followed by slower conversion totrans-11-C 18:1 (FIG. 1). Some of the CLA formed in the rumen isabsorbed into blood and incorporated into milk fat. In addition to thebiohydrogenation reaction leading to CLA synthesis, CLA can also beproduced endogenously from trans-11-C 18:1 in mammary tissues.Trans-11-C 18:1 accumulates in the rumen due to the slower conversionstep to stearic acid, and following absorption from the digestive tractis utilized by different tissues e.g. the mammary gland as a substratefor CLA synthesis by the action of Δ-9-desaturase. Indeed, this may bethe major pathway of CLA synthesis in lactating cows, accounting forabout 64% of the CLA in milk fat.

In addition member of strains of propionibacteria were previouslyidentified to synthesise CLA from linoleic acid by Jiang et al. (1998).

WO 99/29886 describes the use of certain bacterial strains found amongfood grade bacteria, particularly among dairy starter cultures, whichhave the ability to produce CLA in vitro by fermentation. Furthermore WO99/29886 decribes that said bacteria may be used to provide food or feedproducts enriched in CLA, and also pharmaceutical products containingCLA as active ingredients.

Isomerases are enzymes which bring about an isomerisation of substrate.Linoleic acid isomerase catalyzes the isomerisation reaction oflionoleic acid to cis-9, trans-11-octadecadienoic acid. This membranebound enzyme was isolated and characterised by Kepler & Tove (1969). Theisomerisation reaction occurs in the middle of a long hydrocarbon chainremote from any functional group and requires no cofactors. The enzymeexhibits max activity with substrates linoleic and linolenic acid withina narrow concentration range. Three parameters are involved in thebinding of substrate to linoleic acid isomerase: 1.) the π system of asubstrate double bond, 2.) hydrophobic interaction and 3.) hydrogenbonding of the substrate carboxyl group. A proposed model for theisomerization of linoleic acid by linoleic acid isomerase is illustratedin FIG. 2. Pictured at the active site are an electrophile (E) thatinteracts with one of the substrate double bonds, and two basic centers,one of wich (B) is hydrogen bonded to the carboxyl group and the other(B—H) which serves as a donor for the hydrogen added at C-13 (FIG. 2).

WO 99/32604 describes a linoleate isomerase from Lactobacillus reuteri.The enzyme activity leads to the conversion of linoleic acid to sixdifferent CLA species which are as follows: (cis,trans)-9,11-CLA,(trans,cis)-10,12-CLA, (cis,cis)-9,11-CLA, (cis,cis)-10,12-CLA,(trans,trans)-9,11-CLA and (trans,trans)-10,12-CLA.

The disadvantages of the abovementioned process is that the yield of thereaction is very low, the purity of the CLA produced is for anindustrial process not sufficient and that the process takes place withonly low space-time yields. This leads to economically unattractiveprocesses.

Thus, there is still a great need for a single, economicbirthdenological industrial process for the production of CLA which doesnot have the abovementioned disadvantages and therefore for new geneswhich encode enzymes which participate in the biosynthesis of conjugatedlinoleic acid and which allow to synthesize and produce it on anindustrial scale.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts the biohydrogenation of linoleic acid in the rumen.

FIG. 2 depicts the proposed model for the isomerization of linoleic acidby linoleic acid isomerase.

FIG. 3 depicts the method for chromosomal walking by inverse PCR.

FIG. 4 depicts a map of the pCR®2.1-TOPO® vector.

FIG. 5 graphically depicts the fatty acid composition of supernatantfollowing incubation in MRS medium containing 0.5 mg/ml linoleic acidwith B. breve 2258 for 24 hours as compared to the control of B. breve2258 incubated in MRS medium alone.

FIG. 6 depicts a GLC chromatogram of B. breve 2258, with a controlsupernatant.

FIG. 7 depicts a GLC chromatogram of B. breve 2258, with a linoleic acidsupernatant.

FIG. 8 depicts a chromatogram of conjugated linoleic acid standard (Nu-Chek- Prep. Inc, Elysian, Minn.). Separation was performed on ChrompackCP Sil 88 Column (Chrompack, Middleburg, The Netherlands) (60 m×0.25 mmi.d., 0.20 m film thickness). The retention time on this column isdifferent from the column used for the bacterial fatty acids.

FIG. 9 graphically depicts the fatty acid composition of pelletsfollowing incubation in MRS medium containing 0.5 mg/ml linoleic acidwith B. breve for 24 hours. The control was B. breve 2258 icubated inMRS medium alone.

FIG. 10 graphically depicts the fatty acid composition of supernatantfollowing incubation in MRS medium containing 0.5 mg/ml cis-9, trans-11CLA with B. breve 2258 for 48 hours. The control was B. breve 2258incubated in MRS medium alone.

FIG. 11 depicts a GLC chromatogram of B. breve 2258 with conjugatedlinoleic acid as supernatant.

FIG. 12 graphically depicts the fatty acid composition of pelletsfollowing incubation in MRS medium containing 0.5 mg/ml cis-9, trans-11conjugated linoleic acid with B. breve 2258 for 48 hours. The controlwas B. breve 2258 incubated in MRS medium alone.

DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide other isomerases forthe synthesis of unsaturated conjugated fatty acids.

We have found that this object is achieved by an isolated nucleic acidsequence which encodes a polypeptide with conjugated linoleic acidisomerase activity, selected from the following group:

-   a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,-   b) nucleic acid sequences which, as a result of the degeneracy of    the genetic code, are derived from the nucleic acid sequence shown    in SEQ ID NO: 1,-   c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1    which encode polypeptides with the amino acid sequences shown in SEQ    ID NO: 2 and which have at least 75% identity at amino acid level    without substantially reducing the enzymatic activity of the    polypeptides.

These conjugated linoleic acid isomerases can be found in organisms,advantageously microorganisms such as bacteria. The enzyme or theenzymes have a high enzymatic activity for the hydrolytic conversion oflinoleic acid into conjugated linoleic acid.

A derivative (or derivatives) is/are to be understood as meaning, forexample, functional homologs of the enzyme encoded by SEQ ID NO: 1 orits enzymatic activity, viz. enzymes which catalyze the same enzymaticreactions as the enzyme encoded by SEQ ID NO:1. These genes also allowan advantageous preparation of unsaturated conjugated fatty acidspreferably conjugated linoleic acid. Unsaturated fatty acids are to beunderstood, in the following text, as meaning polyunsaturated fattyacids whose double bonds may be conjugated or not conjugated. Thesequence given in SEQ ID NO:1 encodes a novel, unknown isomerase whichparticipates in the synthesis of conjugated linoleic acid in the genusBifidobacterium especially Bifidobacterium breve. The enzyme converts(9Z,12Z)octadecadienoic/linoleic acid to (cis-9,trans-11)octadecaconjudienoic/conjugate linoleic acid. This is termed conjugatedlinoleic acid isomerase or in short terms isomerase hereinbelow.

The nucleic acid sequences according to the invention can in principlebe identified and isolated from all organisms. SEQ ID NO: 1 or itshomologs can advantageously be isolated from fungi, yeasts or bacteria.Bacteria which may be mentioned are Gram-negative and Gram-positivebacteria. The nucleic acid(s) [the plural and singular are intended tohave the same meaning for the application] according to the inventionare preferably isolated by methods known to the skilled worker fromGram-positive bacteria such as Propionibacterium, Lactococcus,Bifidobacterium or Lactobacillus, advantageously from Bifidobacterium.

The nucleic acid sequence according to the invention or its fragmentscan be used advantageously for isolating further genomic sequences bymeans of homology screening.

The abovementioned derivatives can be isolated, for example, from othermicroorganisms such as rumen or intestine bacteria such as Butyrivibrio,Propionibacterium or bacteria which can be isolated for example fromdairy products such as Lactococcus or Lactobacillus.

Such microorganism and the ability of certain rumen-derived strains,including Butyrivibrio fibrisolvens to form CLA from dietary linoleicacid are decribed by Kepler and Tove [The Journal of BiologicalChemistry, Vol.246 No 14: 3612–3620 (1970)], it has also been shown thatcertain cultures used in food fermentations possess the ability togenerate cis-9, trans-11 CLA. Strains of the intestinal flora in rats(Chin et al., J. Nutr. 124, 1993: 694–701), two strains ofPropionibacterium freudenreichii spp. freudenreichii and one strain ofP. freudenreichii subsp. shermanii (Jiang et al., J. Appl. Microbiol.,85, 1998: 95–102), and six lactic cultures, including L. acidophilus(Lin et al., 1999) have been shown to possess this capability. In thisstudy, we assessed a collection of strains, many which are humanintestinal isolates (previously isolated from the human GIT) withprobiotic potential, for ability to form the cis-9, trans-11 CLA isomer,using linoleic acid as the substrate.

Derivatives or functional derivatives of the sequence given in SEQ IDNo.1 are furthermore to be understood as meaning, for example, allelicvariants which have at least 75% homology (=identity) at the derivedamino acid level, preferably at least 80% homology, especiallypreferably at least 85% homology, very especially preferably 90%homology, most preferably 95%, 96%, 97%, 98% or 99% homology. Thehomology (=identity) was calculated over the entire amino acid range.The program used was PileUp (J. Mol. Evolution., 25 (1987), 351–360,Higgins et al., CABIOS, 5 1989: 151–153). The amino acid sequencederived from the abovementioned nucleic acid can be seen from thesequence SEQ ID NO: 2. Allelic variants encompass, in particular,functional variants which can be obtained from the sequence shown in SEQID NO: 1 by means of deletion, insertion or substitution of nucleotides,the enzymatic activity of the derived synthetic proteins being retained.

Such DNA sequences can be isolated from other microorganism as mentionedabove, starting from the DNA sequence described in SEQ ID NO: 1 or partsof these sequences, for example using customary hybridization methods orthe PCR technique. These DNA sequences hybridize with the sequencesmentioned under standard conditions. It is advantageous to use, for thehybridization, short oligonucleotides, for example from the conservedregions, which can be determined by the skilled worker by comparisonwith other known isomerase genes.

Alternatively, it is possible to use longer fragments of the nucleicacids according to the invention or the full sequences for thehybridization. Depending on which nucleic acid: oligonucleotide, longerfragment or full sequence, or depending on which nucleic acid type, viz.DNA or RNA, is used for the hybridization, these standard conditionsvary. Thus, for example, the melt temperatures for DNA:DNA hybrids areapproximately 10° C. lower than those of equally long DNA:RNA hybrids.

Depending on the nucleic acid, standard conditions are understood asmeaning, for example, temperatures between 42 and 58° C. in an aqueousbuffer solution with a concentration of between 0.1 and 5×SSC(1×SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in thepresence of 50% formamide such as, for example, 42° C. in 5×SSC, 50%formamide. The hybridization conditions for DNA:DNA hybrids areadvantageously 0.1×SSC and temperatures between approximately 20° C. and45° C., preferably between approximately 30° C. and 45° C. Thehybridization conditions for DNA:RNA hybrids are advantageously 0.1×SSCand temperatures between approximately 30° C. and 55° C., preferablybetween approximately 45° C. and 55° C. These temperatures which areindicated for the hybridization are examples of calculated melting pointdata for a nucleic acid with a length of approx. 100 nucleotides and aG+C content of 50% in the absence of formamide. The experimentalconditions for the DNA hybridization are described in relevant geneticstextbooks such as, for example, by Sambrook et al., “Molecular Cloning”,Cold Spring Harbor Laboratory, 1989 and can be calculated using formulaeknown to the skilled worker, for example as a function of the length ofthe nucleic acids, the type of hybrid or the G+C content. The skilledworker can find further information on hybridization in the followingtextbooks: Ausubel et al. (eds), 1985, Current Protocols in MolecularBiology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985,Nucleic Acids Hybridization: A Practical Approach, IRL Press at OxfordUniversity Press, Oxford; Brown (ed), 1991, Essential Molecular Biology:A Practical Approach, IRL Press at Oxford University Press, Oxford.

Derivatives are furthermore to be understood as meaning homologs of thesequence SEQ ID NO: 1, for example insect homologs, truncated sequences,simplex DNA of the coding and noncoding DNA sequence or RNA of thecoding and noncoding DNA sequence.

Homologs of the sequence SEQ ID NO: 1 are also to be understood asmeaning derivatives such as, for example, promoter variants. Thesevariants can be altered by one or more nucleotide exchanges, byinsertion(s) and/or deletion(s), without, however, adversely affectingthe functionality or efficacy of the promoters. Moreover, it is possibleto increase the efficacy of the promoters by altering their sequence orto exchange them completely by more efficient promoters from otherorganisms, including other species.

Derivatives are also advantageously to be understood as meaning variantswhose nucleotide sequence in the region −1 to −2000 upstream of thestart codon was altered in such a way that gene expression and/orprotein expression is altered, preferably increased. Moreover,derivatives are also to be understood as meaning variants whose 3′ endwas altered.

To achieve optimal expression of heterologous genes in organisms, it isadvantageous to alter the nucleic acid sequences in accordance with thespecific codon usage used in the organism. The codon usage can bedetermined readily by using computer evaluations of other, known genesof the organism in question.

The amino acid sequences according to the invention are to be understoodas meanings which contain an amino acid sequence shown in SEQ ID NO: 2or a sequence obtainable therefrom by the substitution, inversion,insertion or deletion of one or more amino acid residues, the enzymaticactivity of the protein shown in SEQ ID NO: 2 being retained or notreduced substantially. The term not reduced substantially is to beunderstood as meaning all enzymes which still have at least 10%,preferably 20%, especially preferably 30% of the enzymatic activity ofthe starting enzyme. For example, certain amino acids may be replaced byothers with similar physico-chemical properties (spatial dimension,basicity, hydrophobicity and the like). For example, arginine residuesare exchanged for lysine residues, valine residues for isoleucineresidues or aspartic acid residues for glutamic acid residues.Alternatively, it is possible to exchange the sequence of, add or removeone or more amino acids, or two or more of these measures may becombined with each other.

The nucleic acid construct or nucleic acid fragment according to theinvention is to be understood as meaning the sequence given in SEQ IDNO: 1, sequences which are the result of the genetic code and/or theirfunctional or nonfunctional derivatives, all of which have been linkedfunctionally to one or more regulatory signals, advantageously forincreasing gene expression. These regulatory sequences are, for example,sequences to which inductors or repressors bind and thus regulate theexpression of the nucleic acid. In addition to these novel regulatorysequences, or instead of these sequences, the natural regulation ofthese sequences upstream of the actual structural genes may still bepresent and, if desired, may have been genetically altered in such a waythat the natural regulation has been switched off and the expression ofthe genes increased. However, the expression of the gene construct mayalso have a simpler structure, viz. no additional regulatory signalshave been inserted upstream of the sequence or its derivatives and thenatural promoter with its regulation has not been removed. Instead, thenatural regulatory sequence has been mutated in such a way thatregulation no longer takes place and gene expression is increased. Thesealtered promoters may also be placed upstream of the natural gene ontheir own, in order to increase activity. In addition, the geneconstruct can also advantageously contain one or more so-called enhancersequences functionally linked to the promoter, and these allow anincreased expression of the nucleic acid sequence. It is also possibleto insert, at the 3′ end of the DNA sequences, additional advantageoussequences such as further regulatory elements or terminators. One ormore copies of the conjugated linoleic acid isomerase gene may becontained in the gene construct.

Advantageous regulatory sequences for the process according to theinvention are contained, for example, in promoters such as cos, tac,trp, tet, trp-tet, lpp, lac, lpp-lac, lacI^(q), T7, T5, T3, gal, trc,ara, SP6, λ-P_(R) or in the λ-P_(L) promoter, all of which areadvantageously used in Gram-negative bacteria. Other advantageousregulatory sequences are contained, for example, in the Gram-positivepromoters amy and SPO2, in the yeast or fungal promoters ADC1, MFα, AC,P-60, CYC1, GAPDH, TEF, rp28, ADH.

In principle, all natural promoters with their regulatory sequences asthose mentioned above may be used for the process according to theinvention. In addition, synthetic promoters may also advantageously beused.

The nucleic acid construct advantageously contains, for expression ofthe genes present, in addition 3′ and/or 5′ terminal regulatorysequences to increase expression, these being selected for optimalexpression depending on the selected host organism and gene or genes.

These regulatory sequences are intended to make specific expression ofthe genes and protein expression possible. This may mean, for exampledepending on the host organism, that the gene is expressed oroverexpressed only after induction, or that it is expressed and/oroverexpressed immediately.

The regulatory sequences or factors may for this purpose preferably havea beneficial effect on expression of the introduced genes, and thusincrease it. Thus, an enhancement of the regulatory elements canadvantageously take place at the level of transcription, by using strongtranscription signals such as promoters and/or enhancers. However, it isalso possible to enhance translation by, for example, improving thestability of the mRNA.

The nucleic acid construct (=gene construct, nucleic acid construct,nucleic acid fragment) may also contain further genes to be introducedinto organisms. These genes can be under separate regulation or underthe same regulatory region as the isomerase gene according to theinvention. These genes are, for example, other biosynthesis genes,advantageously of the fatty acid and lipid biosynthesis, which allowincreased synthesis of the isomerase starting material such as linoleicacid.

For optimal expression of heterologous genes in organisms it isadvantageous to modify the nucleic acid sequences in accordance with thespecific codon usage of the organism. The codon usage can easily beestablished on the basis of computer analyses of other, known genes ofthe relevant organism.

For expression in a host organism, for example a microorganism such asfungi or bacteria, the nucleic acid fragment is advantageously insertedinto a vector such as, for example, a plasmid, a phage or other DNA,which vector allows optimal expression of the genes in the host.Examples of suitable plasmids are, in E. coli, pLG338, pACYC184, pBR322,pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290,pIN-III¹¹³-B1, λgt11 or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702.orpIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 orpAJ667, in fungi pALS1, pIL2 or pBB116, in yeasts 2 μM, pAG-1, YEp6,YEp13 or pEMBLYe23, or derivatives of the abovementioned plasmids. Theplasmids mentioned represent a small selection of the plasmids which arepossible. Other plasmids are well known to the skilled worker and can befound, for example, in the book Cloning Vectors (Eds. Pouwels P. H. etal. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).Suitable plant vectors are described, inter alia, in “Methods in PlantMolecular Biology and Biotechnology” (CRC Press), Chapter 6/7, pp.71–119.

In principle all organism are useful as host for the inventive processsuch as fungi, bacteria, yeasts, animals or plants.

In addition to plasmids, vectors are also to be understood as meaningall the other vectors which are known to the skilled worker, such as,for example, phages, viruses such as SV40, CMV, baculovirus, adenovirus,transposons, IS elements, phasmids, phagemids, cosmids, linear orcircular DNA. These vectors can be replicated autonomously in the hostorganism or replicated chromosomally. Autonomous replication ispreferred.

The vector advantageously contains at least one copy of the nucleic acidsequence according to the invention and/or of the nucleic acid fragmentaccording to the invention.

To increase the gene copy number, the nucleic acid sequences orhomologous genes can be introduced, for example, into a nucleic acidfragment or into a vector which preferably contains the regulatory genesequences assigned to the genes in question, or analogously actingpromoter activity. Regulatory sequences which are used in particular arethose which increase gene expression.

To express the other genes contained, the nucleic acid fragmentadvantageously additionally contains 3′- and/or 5′-terminal regulatorysequences to increase expression, these sequences being selected foroptimal expression, depending on the host organism chosen and the geneor genes.

These regulatory sequences should allow the targeted expression of thegenes and protein expression. Depending on the host organism, this maymean, for example, that the gene is expressed and/or overexpressed onlyafter induction, or that it is expressed and/or overexpressedimmediately.

The regulatory sequences or factors can preferably have a positiveeffect on, and thus increase, the gene expression of the genesintroduced. Thus, strengthening of the regulatory elements canadvantageously take place at the transcriptional level by using strongtranscription signals such as promoters and/or enhancers. In, addition,however, strengthening of translation is also possible, for example byimproving mRNA stability.

In a further embodiment of the vector, the gene construct according tothe invention can advantageously also be introduced into the organismsin the form of a linear DNA and integrated into the genome of the hostorganism by means of heterologous or homologous recombination. Thislinear DNA may consist of a linearized plasmid or only of the nucleicacid fragment as vector or of the nucleic acid sequence according to theinvention.

The nucleic acid sequence according to the invention is advantageouslycloned into a nucleic acid construct together with at least one reportergene, and the nucleic acid construct is introduced into the genome. Thisreporter gene should allow easy detectability via a growth assay, afluorescence assay, a chemo assay, a bioluminescence assay or aresistance assay, or via a photometric measurement. Examples of reportergenes which may be mentioned are genes for resistance to antibiotics orherbicides, hydrolase genes, fluorescence protein genes, bioluminescencegenes, sugar metabolism genes or nucleotide metabolism genes, orbiosynthesis genes such as the Ura3 gene, the Ilv2 gene, the luciferasegene, the β-galactosidase gene, the gfp gene, the2-deoxyglucose-6-phosphate phosphatase gene, the β-glucuronidase gene,the β-lactamase gene, the neomycin phosphotransferase gene, thehygromycin phosphotransferase gene or the BASTA (=gluphosinate)resistance gene. These genes allow the transcriptional activity, andthus gene expression, to be measured and quantified easily. In this way,genome sites which show different productivity can be identified.

In a further advantageous embodiment, the nucleic acid sequenceaccording to the invention may also be introduced into an organism onits own.

If it is intended to introduce, into the organism, other genes inaddition to the nucleic acid sequence according to the invention, allcan be introduced into the organism in a single vector with a reportergene, or each individual gene with a reporter gene per vector, it beingpossible for the various vectors to be introduced simultaneously or insuccession.

The host organism (=transgenic organism) advantageously contains atleast one copy of the nucleic acid according to the invention and/or ofthe nucleic acid construct according to the invention.

In principle, the nucleic acid according to the invention, the nucleicacid construct or the vector can be introduced into organisms, forexample plants, by all methods known to the skilled worker.

In the case of microorganisms, the skilled worker can find suitablemethods in the textbooks by Sambrook, J. et al. (1989) Molecularcloning: A laboratory manual, Cold Spring Harbor Laboratory Press, by F.M. Ausubel et al. (1994) Current protocols in molecular biology, JohnWiley and Sons, by D. M. Glover et al., DNA Cloning Vol.1, (1995), IRLPress (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in YeastGenetics, Cold Spring Harbor Laboratory Press or by Guthrie et al. Guideto Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994,Academic Press.

Suitable organisms or host organisms (transgenic organism) for thenucleic acid according to the invention, the nucleic acid construct orthe vector are, in principle, all organisms which are capable ofsynthesizing unsaturated fatty acids, and which are suitable for theexpression of recombinant genes. Examples which may be mentioned aretransgenic plants, transgenic microorganisms such as fungi, for examplethe genus Mortierella, Saprolegnia or Pythium, transgenic bacteria suchas the genus Escherichia, Bifidobacterium, Brevibacterium orCorynebacterium or yeasts such as the genus Saccharomyces. Preferredorganisms are those which are naturally capable of synthesizing oils insubstantial amounts, like fungi such as Mortierella alpina, Pythiuminsidiosum or plants such as soya, oilseed rape, flax, coconut palms,oil palms, safflower or sunflowers, or yeasts such as Saccharomycescerevisiae, with soya, oilseed rape, flax, sunflowers, fungi such asMortierella or bactria such as the genus Bifidobacterium, Brevibacteriumor Corynebacterium being especially preferred. In principle, transgenicanimals, for example Caenorhabditis elegans, are also suitable as hostorganisms.

Advantageously the least organism should grow in the precense of morethan 0.5 mg/ml linoleic acid, preferably more than 1 mg/ml, morepreferably more than 1.5 mg/ml and most preferably more than 2 mg/mllinoleic acid. The skilled worker knows how to identify such preferedorganism by using a simple gows assay.

With regard to the nucleic acid sequence as depicted in SEQ ID NO: 1, anucleic acid construct which contains said nucleic acid sequence or anorganism (=transgenic organism) which is transformed with said nucleicacid sequence or said nucleic acid construct, “transgene” means allthose constructs which have been brought about by genetic manipulationmethods and in which either

-   a) the nucleic acid sequence as depicted in SEQ ID NO: 1 or a    derivative thereof, or-   b) a genetic regulatory element, for example a promoter, which is    functionally linked to the nucleic acid sequence as depicted in SEQ    ID NO: 1 or a derivative thereof, or-   c) (a) and (b) is/are not present in its/their natural genetic    environment or has/have been modified by means of genetic    manipulation methods, it being possible for the modification to be,    by way of example, a substitution, addition, deletion, inversion or    insertion of one or more nucleotide radicals. “Natural genetic    environment” means the natural chromosomal locus in the organism of    origin or the presence in a genomic library. In the case of a    genomic library, the natural, genetic environment of the nucleic    acid sequence is preferably at least partially still preserved. The    environment flanks the nucleic acid sequence at least on one side    and has a sequence length of at least 50 bp, preferably at least 500    bp, particularly preferably at least 1000 bp, very particularly    preferably at least 5000 bp.

The use of the nucleic acid sequence according to the invention or ofthe nucleic acid construct according to the invention for the generationof transgenic plants is therefore also subject matter of the invention.

In the conversion with the enzyme according to the invention, one doublebond is shifted so that the double bonds which participate in thereaction are conjugated (FIG. 2).

The enzyme (=conjugated linoleic isomerase) advantageously catalyzes theconversion of linoleic acid (18:2, 9Z,12Z) to conjugated cis-9, trans-11linoleic acid.

The invention furthermore relates to a process for the production ofconjugated unsaturated fatty acids especially conjugated linoleic acid,which comprises introducing at least one above-described nucleic acidsequence according to the invention or at least one nucleic acidconstruct according to the invention into a preferentially oil-producingorganism, growing this organism, isolating the oil contained in theorganism and liberating the fatty acids contained in the oil.

The invention also includes a process for the production oftriglycerides with an increased content of conjugated unsaturated fattyacids especially conjugated linoleic acid, which comprises introducingat least one above-described nucleic acid sequence according to theinvention or at least one nucleic acid construct according to theinvention into a preferentially oil-producing organism, growing thisorganism and isolating the oil contained in the organism.

Both processes advantageously allow the synthesis of fatty acids oftriglycerides with an increased content of unsaturated fatty acids suchas conjugated linoleic acid.

The host organisms advantageously contain 0.5 U/g DBM (=dry bio-mass)CLA isomerase activity, preferably 4 U/g DBM, particularly preferably20–150 U/g DBM, very particularly preferably 40–150 U/g DBM.

The process according to the invention is advantageously carried out ata temperature between 0° C. and 95° C., preferably between 10° C. and85° C., particularly preferably between 15° C. and 75° C., mostpreferably between 20° C. and 60° C.

The pH in the process (in vitro) according to the invention isadvantageously kept between pH 4 and 12, preferably between pH 6 and 9,particularly preferably between pH 6 and 8, very particularly preferablybetween pH 6.0 and 7.5.

The purities of the different CLA isomers which are produced in theinventive process is of at least 70%, preferably of at least 80%,particularly preferably of at least 90%, very particularly preferably atleast 98%.

It is possible to use for the process according to the invention growingcells which comprise the nucleic acids, nucleic acid constructs orvectors according to the invention. It is also possible to use restingor disrupted cells. Disrupted cells mean, for example, cells which havebeen made permeable by treatment with, for example, solvents, or cellswhich have been ruptured by an enzyme treatment, by a mechanicaltreatment (for example French press or ultrasound) or by another method.The crude extracts obtained in this way are advantageously suitable forthe process according to the invention. Purified or partially purifiedenzymes can also be used for the process. Likewise suitable areimmobilized microorganisms or enzymes which can advantageously be usedin the reaction.

If free organisms or enzymes are used for the process according to theinvention, these are expediently removed, for example by filtration orcentrifugation, before the extraction. It is advantageous that this isunnecessary on use of immobilized organisms or enzymes, but it may stilltake place.

With the types of work up mentioned, the product of the process(=conjugated unsaturated fatty acids, especially CLA preferably. 9-cis,11-trans CLA) according to the invention can be isolated in yields offrom 20 to 100%, preferably from 30 to 100%, particularly preferablyfrom 50 to 100%, more particularly preferably from 60 to 100%, 70 to100%, 80 to 100%, 90 to 100%, based on the amount of linoleic acidemployed for the reaction. In addition, the products have a highisomeric purity, which can advantageously be further increased wherenecessary by the crystallization. The inventive process leads to cis-9,trans-11 octadecadienoic acid as major product.

Linoleic acid as a major starting material can be added to the reactionmixture batchwise, semibatchwise or continuously.

The process according to the invention can be carried out in vivo or invitro batchwise, semibatchwise or continuously.

The concentration of the starting material for the process which ispreferably linoleic acid is higher than 0,5 mg/ml, preferably higherthan 2 mg/ml, more preferably higher than 3 mg/ml.

The concentration of CLA as product of the inventive process in theculture medium is higher than 1 mg/ml, preferably higher than 2 mg/ml,more preferably higher than 3 mg/ml.

The products obtained in this way are suitable as starting material forthe synthesis of mono-, di- or triglycerols and derivatives thereof.These substances and the isomer pure CLA obtained can be used incombination with one another or alone for producing drugs, foodstuffs,animal feeds or cosmetics.

Examples of organisms for the abovementioned processes are plants suchas Arabidopsis, soya, peanuts, castor, sunflowers, corn, cotton, flax,oilseed rape, coconut palms, oil palms, safflower (Carthamus tinctorius)or cacao, microorganisms such as the fungi Mortierella, Saprolegnia orPythium, bacteria such as gram-positive or gram-negative bacteria of thegenera Escherichia, Lactobacillus, Lactococcus, Propionibacterium,Bifidobacterium, Brevibacterium, Corynebacterium, Pediococcus orButyrivibrio, yeasts such as the genus Saccharomyces. Preferredorganisms are those which can naturally synthesize oils in substantialamounts, such as fungi, for example Mortierella alpina, Pythiuminsidiosum, or plants such as soya, oilseed rape, flax, coconut palms,oil palms, safflower, castor, peanuts, cacao or sunflowers, or yeastssuch as Saccharomyces cerevisiae or bacteria such as Propionibacteriumfreudenreichii, Propionibacterium acidipropionici, Propionibacteriumacnes, Propionibacterium avidum, Propionibacterium granulosum,Propionibacterium jensenii, Propionibacterium lymphophilum,Propionibacterium propionicum, Propionibacterium theonii, Pediococcusacidilactici, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcushalophilus, Pediococcus parvulus, Pediococcus pentosaceus,Bifidobacterium breve, Bifidobacterium dentium, Bifidobacteriumadolescentis, Bifidobacterium longum, Bifidobacterium angulatum,Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacteriumasteroides, Bifidobacterium boum, Bifidobacterium catenulatum,Bifidobacterium choerinum, Bifidobacterium coryneforme, Bifidobacteriumcuniculi, Bifidobacterium gallinarum, Bifidobacterium globosum,Bifidobacterium indicum, Bifidobacterium infantis, Bifidobacteriummagnum, Bifidobacterium merycicum, Bifidobacterium minimum,Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum,Bifidobacterium pullorum, Bifidobacterium subtile, Bifidobacterium suis,Bifidobacterium thermophilum, Butyrivibrio fibrisolvens, Butyrivibriocrossotus, Lactobacillus acidophilus, Lactobacillus acetotolerans,Lactobacillus agilis, Lactobacillus alimentarius, Lactobacillusamylovorus, Lactobacillus animalis, Lactobacillus aviarius,Lactobacillus bifermentans, Lactobacillus bifidus, Lactobacillus brevis,Lactobacillus buchneri, Lactobacillus carnis, Lactobacillus casei,Lactobacillus catenaformis, Lactobacillus collinoides, Lactobacillusconfusus, Lactobacillus coryniformis, Lactobacillus crispatus,Lactobacillus delbrueckii, Lactobacillus divergens, Lactobacillusfarciminis, Lactobacillus fermentum, Lactobacillus fructivorans,Lactobacillus fructosus, Lactobacillus gasseri, Lactobacillusholotolerans, Lactobacillus hamsteri, Lactobacillus helveticus,Lactobacillus kefir, Lactobacillus lactis, Lactobacillus malefermentans,Lactobacillus mali, Lactobacillus minor, Lactobacillus minutus,Lactobacillus parabuchneri, Lactobacillus paracasei, Lactobacilluspentoaceticus, Lactobacillus plantarum, Lactobacillus reuteri,Lactobacillus ruminis, Lactobacillus sake, Lactobacillus salivarius,Lactobacillus xylosus, Lactococcus garviae, Lactococcus lactis,Lactococcus plantarum, Lactococcus raffinolactis, Corynebacteriumaccolens, Corynebacterium acetoacidophilum, Corynebacteriumacetoglutamicum, Corynebacterium acnes, Corynebacterium alkanolyticum,Corynebacterium alkanum, Corynebacterium ammoniagenes, Corynebacteriumamycolatum, Corynebacterium aquaticum, Corynebacterium aurantiacum,Corynebacterium barkeri, Corynebacterium callunae, Corynebacteriumcystitidis, Corynebacterium dioxydans, Corynebacterium equi,Corynebacterium flaccumfaciens, Corynebacterium flavescens,Corynebacterium fujiokense, Corynebacterium glutamicum, Corynebacteriumglycinophilum, Corynebacterium haemolyticum, Corynebacterium herculis,Corynebacterium histidiolovorans, Corynebacterium hoagii,Corynebacterium humiferum, Corynebacterium hydrocarboclastum,Corynebacterium hydrocarbooxydans, Corynebacterium insidiosum,Corynebacterium jeikeium, Corynebacterium kutscheri, Corynebacteriumlilium, Corynebacterium liquefaciens, Corynebacterium matruchotit,Corynebacterium mediolanum, Corynebacterium melassecola, Corynebacteriumminutissimum, Corynebacterium mycetoides, Corynebacterium nephridii,Corynebacterium nitrophilus, Corynebacterium paraldehydium,Corynebacterium paurometabolum, Corynebacterium petophilum,Corynebacterium pilosum, Corynebacterium primorioxydans, Corynebacteriumrubrum, Corynebacterium simplex, Corynebacterium striatum,Corynebacterium tuberculostrearicum, Corynebacterium variabilis,Corynebacterium vitarumen, Corynebacterium xerosis, Brevibacteriumacetylicum, Brevibacterium albidum, Brevibacterium album, Brevibacteriumalkanolyticum, Brevibacterium alkanophilum, Brevibacterium ammoniagenes,Brevibacterium butanicum, Brevibacterium casei, Brevibacterium cerinum,Brevibacterium citreum, Brevibacterium divericatum, Brevibacteriumepidermidis, Brevibacterium flavum, Brevibacterium frigoritolerans,Brevibacterium fuscum, Brevibacterium glutamigenes, Brevibacteriumhalotolerans, Brevibacterium healii, Brevibacterium helvolum,Brevibacterium immariohilium, Brevibacterium imperiale, Brevibacteriumincertum, Brevibacterium insectiphilium, Brevibacterium iodinum,Brevibacterium ketoglutamicum, Brevibacterium ketosoreductum,Brevibacterium lactofermentum, Brevibacterium linens, Brevibacteriumluteum, Brevibacterium lyticum, Brevibacterium maris, Brevibacteriumparaffinoliticum, Brevibacterium protophormiae, Brevibacterium pusillum,Brevibacterium roseum, Brevibacterium saccharolyticum, Brevibacteriumsaperdae, Brevibacterium seonmiso, Brevibacterium stationis,Brevibacterium sterolicum, Brevibacterium sulfureum, Brevibacteriumtaipei or Brevibacterium testaceum; or plants such as soya, oilseedrape, flax, sunflowers or Saccharomyces cerevisiae are especiallypreferred, most preferred are Brevibacterium ammoniagenes,Brevibacterium flavum, Brevibacterium ketoglutamicum, Brevibacteriumiodinum, Brevibacterium lactofermentum, Brevibacterium linens,Brevibacterium saccharolyticum, Corynebacterium acetoglutamicum,Corynebacterium ammoniagenes, Corynebacterium glutamicum,Corynebacterium melassecola, Bifidobacterium breve, Bifidobacteriumdentium or Bifidobacterium pseudocatenulatum.

1. Bifidobacterium

The genus Bifidobacterium consists of about 30 species of gram-positive,anaerobic nonmotile, various shaped rods (Jay, 1996). They are non-sporeforming, catalase-negative and non-acid fast. Cells often stainirregularly with methylene blue. Some species can tolerate O₂ in thepresence of CO₂. Optimum growth temperature is 37–41° C. and optimal pHfor initial growth is 6.5–7.0 (Bergey's Manual, 1989). Bifidobacteriaproduce acetic and lactic acid in the molar ratio of 3:2, and also asmall amount of formic acid, ethanol and succinic acid. A uniquecharacteristic of bifidobacteria is the glucose degradation pathway bythe “fructose-6-phosphate shunt” using the enzymefructose-6-phosphoketolase. As a nitrogen source, bifidobacteria canutilize ammonium. The G+C content of the DNA varies from 55–67 mol %.Bifidobacteria were first isolated from human infants' faeces (György,1953), and are predominantly found in the intestines of humans andswine.

2. Propionibacterium

Propionibacterium species are small, pleomorphic rods of 0.5–0.8 m indiameter and 1–5 m in length, often with one end rounded and onepointed. The cells can be coccoid, bifid or branched and they appearsingly, in pairs or in short chains in V or Y configurations or in“Chinese character” arrangements (Bergey's Manual, 1989). This genus areGram-positive, non sporing chemoorganotrophs that are nonmotile. Theyare slow growing, requiring several days of incubation for visible signsof growth. They usually grow better anaerobically than aerobically at anoptimum temperature of 30–37° C. (Cogan and Accolas, 1996). Fermentationproducts are large amounts of propionic and acetic acid, however, theyalso produce CO₂, which is responsible for the large eyes in Swisscheese, formed during lactate fermentation, according to;3 lactate+2 propionate+1 acetate+1CO₂

Propionibacteria are generally catalase-positive. The G+C content oftheir DNA varies from 53–67 mol %. Based on habitats, there are twoprincipial groups of microorganisms, i.e. strains from cheese and dairyproducts and strains found on human skin or in the intestines.

Depending on the host organism, the organisms used in the processes aregrown or cultured in the manner known to those skilled in the art. As arule, microorganisms are grown in a liquid medium which contains acarbon source, usually in the form of sugars, a nitrogen source, usuallyin the form of organic nitrogen sources such as yeast extract or saltssuch as ammonium sulfate, a phosphate source such as potassium hydrogenphosphate, trace elements such as iron salts, manganese salts, magnesiumsalts and, if required, vitamins, at temperatures between 0° C. and 100°C., preferably between 10° C. and 60° C., more preferably between 15° C.and 50° C., while gassing in oxygen. The pH of the liquid medium can bemaintained at a fixed value, i.e. the pH is regulated while culturetakes place. The pH should then be in a range between pH 2 and pH 9.However, the microorganisms may also be cultured without pH regulation.Culturing can be effected by the batch method, the semi-batch method orcontinuously. Nutrients may be supplied at the beginning of thefermentation or fed in semicontinuously or continuously.

Post-transformation, plants are first regenerated and then grown orplanted as usual.

After the organisms have been grown, the lipids are obtained in theusual manner. To this end, the organisms can first be harvested and thendisrupted, or they can be used directly. It is advantageous to extractthe lipids with suitable solvents such as apolar solvents, for examplehexane, or polar solvents, for example ethanol, isopropanol, or mixturessuch as hexane/isopropanol, phenol/chloroform/isoamyl alcohol, attemperatures between 0° C. and 80° C., preferably between 20° C. and 50°C. As a rule, the biomass is extracted with an excess of solvent, forexample with an excess of solvent to biomass of 1:4. The solvent issubsequently removed, for example by distillation. The extraction mayalso be carried out with supercritical CO₂. After the extraction, theremainder of the biomass can be removed, for example, by filtration.Standard methods for the extraction of fatty acids from plants andmicroorganisms are described in Bligh et al. (Can. J. Biochem. Physiol.37, 1959: 911–917) or Vick et al. (Plant Physiol. 69, 1982: 1103–1108).

The crude oil thus obtained can then be purified further, for example byremoving cloudiness by adding polar solvents such as acetone or apolarsolvents such as chloroform, followed by filtration or centrifugation.Further purification via columns or other techniques is also possible.

To obtain the free fatty acids from the triglycerides, the latter arehyrolyzed in the customary manner, for example using NaOH or KOH.

Another aspect of the invention is the production of conjugated linoleicacid by cultivating a microorganism of the genus Bifidobacterium,Corynebacterium or Brevibacterium in the presence of linoleic acid andisolating the formed conjugated linoleic acid.

In addition the invention furthermore relates to the use ofmicroorganism of the genus Bifidobacterium as a probiotic in food andfeed. Such use is in order to prevent or reduce the effects ofdiarrhoea, infections, cancer and antibiotic treatment.

The invention furthermore relates to conjugated unsaturated fatty acidsand triglycerides with an increased content of conjugated unsaturatedfatty acids which have been prepared by the abovementioned processes,and to their use for the preparation of foodstuffs, animal feed,cosmetics or pharmaceuticals. To this end, they are added to thefoodstuffs, animal feed, cosmetics or pharmaceuticals in the customaryquantities.

The invention is illustrated in greater detail in the examples whichfollow:

EXAMPLES

Nineteen strains of Lactobacillus, 2 strains of Lactococcus, 1 strain ofPediococcus, 4 strains of Propionibacterium and 23 strains ofBifidobacterium were screened for their ability to produce conjugatedlinoleic acid (CLA) from linoleic acid. Of these, 7 strains ofBifidobacterium, as well as 2 strains of Propionibacterium produced thecis-9, trans-11 CLA isomer from linoleic acid. In contrast, strains usedof Lactobacillus, Lactococcus and Pediococcus lacked the ability tosynthesise CLA. CLA (cis-9, trans-11 isomer) production by the genusBifidobacterium was shown to exhibit considerable interspeciesvariation, with B.breve and B.dentium being the most efficient producersamong the strains tested, yielding up to 65% conversion of linoleic acidto CLA at linoleic acid concentrations of 0.2–1.0 mg/ml in MRS medium.The growth of B.breve strains was inhibited by increasing concentrationsof linoleic acid. Viability of B.breve 2257 was unaffected in thepresence of up to 0.5 mg/ml linoleic acid for 48 h but was dramaticallyreduced to 1.5% survival at 1 mg/ml linoleic acid. However, viability ofthe B.breve strains NCFB2258, NCTC 11815, NCIMB 8815 and NCIMB 8807 wasreduced to <60% at linoleic acid concentrations of 0.2 mg/ml. These datasuggest that certain strains of bifidobacteria may have applications toelevate CLA content of food products and CLA status in humans.

Materials and Methods

Example 1

Maintenance of Bacterial Strains

The 19 strains of Lactobacillus, 2 strains of Lactococcus, 1 strain ofPediococcus, 4 strains of Propionibacterium and 19 strains ofBifidobacterium were used in this study.

Table 1 shows an example of the CLM production of certain strainstested.

TABLE 1 Screening of Propionibacterium strains for CLA production fromlinoleic acid in MRS media. Remaining LA CLA produced Strain (μg/ml)(μg/ml) P. acidi propionici 87.5 — NCFB 5633 P. freudenreichii spp. 73.012.1 shermanii LMG 16424 P. freudenreichii spp. 73.0 13.8 shermanii JS(Visby)

The Lactobacilli, Pediococci and Bifidobacterium strains were culturedin MRS (Difco Laboratories, Detroit, Mich., USA) under anaerobicconditions (anaerobic jars with ‘Anaerocult A’ gas packs; Merck,Darmstadt, Germany) and 1.5% (w/v): agar (Oxoid Ltd. Basingstoke,Hampshire, UK) was included for plating. Pediococci, Lb.reuteri NCIMB702655, Lb. reuteri NCIMB 7025656 and Lb. reuteri DSM 20016 wereroutinely cultured at 30° C. and the remaining Lactobacillus strainswere cultured at 37° C. for 24 h. For Bifidobacterium, 0.05% (w/v)L-cysteine hydrochloride (98% pure, Sigma Chemical Co. St. Louis, Mo.,USA) was added to the medium and cultures were grown for 48 h at 37° C.under anaerobic conditions. Lactococcus strains were cultured in MRSunder aerobic conditions at 30° C. for 24 h. The Propionibacteriumstrains were cultured in sodium lactate medium (SLM, Malik et al. 1968)at 20° C. for 72 h under anaerobic conditions. Total viable counts weredetermined by pour plating of 10-fold serial dilutions in MaximumRecovery Diluent (Oxoid), using MRS agar for lactobacilli and MRS agarwith 0.05% (w/v) cysteine for bifidobacteria.

Example 2

Assay for Microbial CLA Production

Prior to examination of the strains for CLA production, each wassubcultured twice in MRS broth (supplemented with cysteine, 0.05% w/vfor Bifidobacterium) for 48 h, using a 1% innoculum. All strains werethen cultured in MRS broth (supplemented with cysteine, 0.05% w/v forBifidobacterium), spiked with different concentrations of free linoleicacid (LA: cis-9, cis-12-octadecadienoic acid, 99% pure, Sigma ChemicalCo.). This was added as a 30 mg/ml stock solution of linoleic acid in 2%(v/v) Tween 80 (polyoxyethylene sorbitan mono-oleate; Merck-Schuchardt,Germany), which was previously sterile-filtered through a 0.45 μmMinisart filter (Sartφrius AG, Germany). The strains were inoculated toa density of 10⁶ cfu/ml in free linoleic acid-containing MRS media andincubated for their respective times and temperatures (described above).Following incubation, 5 ml of the cultures were centrifuged at 960×g for5 min at room temperature (Sanyo MISTRAL 2000R Centrifuge).

The fatty acid composition of the resulting supernatant was analysed asfollows. Initially, C_(13:0) (tridecanoic acid, 99% pure, Sigma ChemicalCo.) was added to 4 ml of the resulting supernatant, as an internalstandard at a concentration of 0.25× the initial linoleic acidconcentration and lipid extraction was performed as follows. Twomilliliters of isopropanol (99% purity, Alkem Chemicals Ltd., Cork,Ireland) was added to the supernatant and the samples were vortexed for30 sec. A total of 4.5 ml hexane (99%. purity, LabScan Ltd., Dublin,Ireland) was added to this and the mixture plased on a shaking platformfor 3 min before centrifugation at 960×g for 5 min at room temperature.The supernatant (the hexane layer containing the lipids) was removed andthe procedure was repeated twice. The hexane layers were pooled andstored at −20° C. prior to preparation of fatty acid methyl esters(FAME) for gas liquid chromatographic (GLC) analysis.

Example 3

Preparation of Fatty Acid Methyl Esters (FAME) and GLC Analysis

The lipid extracts in hexane were analysed by GLC followingacid-catalyzed methylation as described previously (Stanton et al.,1997). Free fatty acids in oils such as sunflower and soybean oils werecalculated as the difference between fatty acid concentrations obtainedfollowing acid and base catalyzed methylation, performed using 2 Nmethanolic KOH (Sigma Chemical Co.) at room temperature.

The GLC was performed with reference to the internal standard C_(13:0).Separation of the FAME was performed on a Chrompack CP Sil 88 column(Chrompack, Middleburg, The Netherlands, 100 m×0.25 mm i.d., 0.20 μmfilm thickness), using helium as carrier gas at a pressure of 37 psi.The injector temperature was held isothermally at 225° C. for 10 min andthe detector temperature was 250° C. The column oven was held at aninitial temperature of 140° C. for 8 min and then programmed at anincrease of 8.5° C./min to a final temperature of 200° C., which washeld for 41 min. Collected data were recorded and analyzed on aMinichrom PC system (VG Data System, Manchester, UK). The cis-9,trans-11 CLA isomer was identified by retention time with reference to aCLA mix (Nu-Chek-Prep. Inc., Elysian, Minn.). The percentage conversionto CLA and the remaining linoleic acid in the broth were calculated bydividing the amount of CLA and linoleic acid present in the broth afterinoculation and incubation with the various cultures used with theamount of linoleic acid present in the spiked broth before incubation.

Example 4

CLA Production by B.Breve NCFB 2258 Using triglyceride Bound LinoleicAcid as Substrate

B.breve NCFB 2258 was further investigated for ability to utilisetriglyceride bound linoleic acid as substrate for CLA production.B.breve NCFB 2258 was inoculated from a fully grown culture into MRSbroth with added cysteine (0.05%) and trilinolein (C_(18:2), cis-9,cis-12, 99% pure, Sigma Chemical Co.), soybean oil and sunflower oil(purchased from a local grocery store) containing known linoleic acidconcentrations. The triglyceride mixtures were sterile-filtered through0.45 μm Minsart filters and added as 5 mg/ml aqueous solutions in 2.5%(v/v) Tween 80. Substantial vortexing was required to dissolve the fatparticles. The volume of the triglyceride stock solutions added wascalculated to give a final concentration of 0.2 mg linoleic acid/ml ofbroth. B.breve 2258 was inoculated into MRS broth in the presence of thetriglyceride substrates under anaerobic conditions at 37° C. andincubated for 48 h.

Additional Examples

Strains used in this study included Bifidobacterium breve 2258, whichwas obtained from NCIMB (National Collection of Industrial and MarineBacteria, Aberdeen, Scotland) and Propionibacterium freudenreichiishermanii 9093 (PFS), which was obtained from Kemikalia AB, Lund,Sweden.

The bifidobacteria strain, B.breve 2258, used in this study was culturedin MRS media (pH 6.0) (Oxoid Ltd, Hampshire, UK) with 1.5% (w/v) agar(Oxoid Ltd, Hampshire, UK) and 0.05% (w/v) L-cysteine hydrocloride(Sigma Chemical, 98% pure) under anaerobic conditions using anaerobicjars with ‘Anaerocult A’ gas packs (Merck, Darmstadt, Germany). Thecultures were grown for 72 h at 37° C. and then subcultured (restrokefrom a singe colony) for pure colonies.

The strain of propionibacteria, P.freudenreichii shermanii, 9093 (PFS),used in this study was cultured in sodium lactate medium (SLM), pH 7.0(Malik et al., 1968) at 30° C. for 72 h under anaerobic conditions andthen subcultured for purity.

Example 5

Screening Assay for Microbial CLA Production from Linoleic Acid

After subculturing twice, strains B.breve 2258 and P. freudenreichiishermanii 9093 were cultured in MRS broth containing 0.05% cysteine(w/v) and SLM, respectively, for 24 h (bifidobacteria) and 48 h(propionibacteria). A 1% (v/v) inoculum was then transferred to newbroth tubes containing 0.5 mg/ml linoleic acid (LA) (Sigma Chemical Co.St. Louis, Mo., USA, 99% pure), added as 30 mg/ml stock solution in 2%(v/v) Tween 80 (polyoxyethylene sorbitan mono-oleate) (Merck-Schuchardt,Germany) which was sterile filtered through 0.45 m Minisart filter(Sartrius AG, Germany) and stored in the dark at −20° C. The cultureswere then grown for 48 h (bifidobacteria) and 72 h (propionibacteria) attheir respective temperatures, prior to lipid extraction of bothsupernatant and bacterial pellets. All extractions were performed induplicate and control cultures were incubated in the absence of addedfatty acids.

Example 6

Screening Assay for Microbial Biohydrogenation of CLA

After subculturing twice, both strains were cultured in their respectivemedia for 24 h (bifidobacteria) and 48 h (propionibacteria), and then a1% (v/v) inoculum was transferred to new broth tubes, containing 0.5mg/ml pure cis-9, trans-11 CLA (Matreya Inc. Pa., USA). The CLA wasadded as a 30 mg/ml stock solution in 2% (v/v) Tween 80, which wassterile filtered. The cultures were grown for 48 h (bifidobacteria) and72 h (propionibacteria) at their respective temperatures, followed bylipid extraction of both supernatant and bacterial pellets. Allextractions were performed in duplicate and control cultures wereincubated in the absence of added fatty acids.

Example 7

Lipid Extraction of Supernatant

After transferring 10 ml of the cultures inoculated with either CLA orLA to 15 ml centrifuge tubes (Sarstedt, Numbrecht, Germany),centrifugation was performed at 2197×g for 20 min at room temperature(20C), using a Sanyo Mistral 2000 R centrifuge. To 4 ml of thesupernatant were added 0.75 mg C 13:0 (tridecenoic acid, Sigma, 99%pure) as internal standard prior to lipid extraction, performed asfollows: 2 ml isopropanol (Alkem Chemicals Ltd. Cork, Ireland, 99%purity) and 1.5 ml hexane (LabScan Ltd. Dublin, Ireland, 99% purity)were added to the supernatant and vortex mixed, and a further 3 ml ofhexane were then added and the mixture, which was vortex mixed againbefore centrifugation at 2197×g for 5 min. All upper layer (hexane layercontaining fatty acids) was transferred to a screw capped glass tube anddried down under N₂ gas stream. Tubes were then stored at −20° C. priorto preparation of fatty acid methyl esters (FAME) for GLC (Gas LiquidChromatography) analysis. Following GLC, results were calculated as mgfatty acid per ml of broth.

Example 8

Lipid Extraction of Pellet

After removal of supernatant, bacterial cells (pellets) from 10 ml ofgrown culture were washed by adding and resuspending them in 1 ml salinesolution (0.137 M NaCl, 7.0 mM K₂HPO₄, 2.5 mM KH₂PO₄) and vortex mixingbefore centrifuging at 3632×g for 30 min. After removal of supernatant,pellets were again resuspended in 1 ml saline solution followed bycentrifugation at 3632×g for 15 min and removal of the supernatantagain. The cells were again resuspended in 1 ml saline solution, towhich was added 0.75 mg C 13:0 (as described above for supernatant) asinternal standard prior to preparation of FAME for GLC analysis.Following GLC, results were calculated as mg fatty acids from 1 ml offully grown culture and expressed as mg fatty acids/ml.

Example 9

Preparation of Fatty Acid Methyl Esters (FAME)

Acid catalyzed methylation, which results in derivatisation of both freefatty acids and triglyceride bound fatty acids was performed asdescribed below: Extracted lipids from supernatants and pellets (asdescribed in sections 2.4.1 and 2.4.2) in screw capped glass tube, wereresuspended in 12 ml, 4% methanolic HCl (v/v) (Supelco Inc. Bellefonte,Pa., USA) in methanol and vortex mixed for 10 sec. The lipids inmethanolic HCl were incubated at 60° C. for 1 h with vortex mixing every10 min. Two ml of water saturated with hexane and 5 ml of hexane werethen added to the solution which was vortex mixed for 30 sec, and thenallowed to stand for 30 min. The clear top layer, containing the FAMEwas subsequently transferred to a tube and 2 ml of water saturated withhexane were added and the solution again vortex mixed and allowed tostand for 30 min. Following this, the top layer was transferred to a newtube and the methylation reaction terminated by addition to this layerof 0.5 g anhydrous sodium sulphate (Sigma, 99% purity) and vortex mixedfor 5 sec. After 1 h, the top layer was removed and stored at −20° C.prior to GLC analysis.

Example 10

GLC Analysis

The free fatty acids were analysed as fatty acid methyl esters (FAME)using a gas liquid chromatograph (GLC-Varian 3400, Varian, Harbor City,Calif., USA) fitted with a flame ionization detector (FID) and a SeptunProgrammable Injector (SPI). Quantification of fatty acids was performedwith reference to the internal standard (C 13:0). Separation of fattyacids was performed on a Chrompack CP Sil 88 column (Chrompack,Middleburg, The Netherlands) (100 m×0.25 mm i.d., 0.20 m filmthickness), using He as carrier gas at a pressure of 33 psi. Theinjector temperature was held isothermally at 225° C. for 10 min and thedetector temperature was 250° C. The column oven was held at an initialtemperature of 140° C. for 8 min, and then programmed at an increase of8.5 C/min to a final temperature of 200° C., which was held for 41 min.

Collected data were recorded and analyzed on a Minichrom PC system (VGData System, Manchester, UK). The cis-9, trans-11 CLA isomer wasidentified by retention time with reference to CLA standards (MatreyaInc. Pa., USA), and trans-11-C 18:1 and stearic acid (Sigma Chemical Co.St. Louis, Mo., USA) identified by reference to their standard fattyacids. To calculate correction factors for the CLA isomer peaks theinternal standard C 13:0 was used using the following formula:Cf_(I)=(A_(IS)×Wt_(I))/(A_(I)×Wt_(IS)), where Cf_(I) is the correctionfactor for the actual CLA isomer, A_(IS) is refers to the area of theinternal standard (C 13:0), A_(I) is the area of the CLA peak, Wt_(I) isthe weight of the CLA isomer and Wt_(IS) refers to the weight of theinternal standard. The quantity of CLA was expressed as mg/ml broth, andthroughout the thesis CLA refers to the cis-9, trans-11 isomer (unlessotherwise stated), which was the most abundant CLA isomer formed duringmicrobial biohydrogenation of free linoleic acid. The response factorsof the individual fatty acids were calculated relative to the area of C18:0, which was assigned a response factor of 1.00. The % conversion toCLA and the % remaining linoleic acid in the broth were calculated bydividing the amount of CLA and linoleic acid present in the broth afterinoculation with the cultures used, with the amount of linoleic acidpresent in the spiked broth before incubation.

Example 11

DNA Sequence Analysis

The sequence encoding the putative linoleic acid isomerase gene fromLactobacillus reuteri (Rosson, et al., 1999, WO 99/32604) was comparedto sequence databases (GenBank+unfinished genomes databases), using theBLAST suite of programs (Altschul et al., 1990). Proteins exhibitingsignificant similarity were aligned using DNAStar software (DNAStar Inc.Madison, Wisc.) and conserved motifs were identified. Degenerateoligonucleotide primers, specific for these motifs, were designed andused in PCR reactions. For each primer, one general and one specificprimer were designed (codon usage of the strains were taken underconsideration when designing the specific primers). Primers weredesigned as follows (all sequences are written 5′–3′):

Primer 1 (amino acid sequence): G N Y E A F A 1a (general): GGI,AA(C/T), TA(C/T), GA(A/G), GCI, TT(T/C), GA(A/G). 1b (specific forbifidobacteria): GGI, AAC, TAC, GAA, GCI, TTC, GAA Primer 2 (amino acidsequence): R G G R E M E N H F E C 2a (general): CGI, GGI, GGI, CGI,GA(A/G), ATG, GA(A/G), AA(C/T), CA(C/T), TT(C/T), GA(A/G), TG(C/T). 2b(spec. for bif.): CGI, GGI, CGI, GAA, ATG, GAA, AAC, CA(C/T), TTC, GAA,TGC. Primer 3 (amino acid sequence): Y W X T M F A F E 3a (general):TA(C/T), TGG, III, ACI, ATG, TT(C/T), GCI, TT(C/T), GA(A/G). 3b (spec,for bif.): TAC, TGG, III, ACC, ATG, TTC, GCI, TTC, GAA. Primer 4 (aminoacid sequence): Y W X T M F A F E 4a (general): TC, (G/A)AA, IGC,(A/G)AA, CAT, IGT, III, CCA, (A/G)TA. 4b (spec. for bif.): TTC, GAA,IGC, GAA, CAT, GGT, III, CCA, GTA. Primer 5 (amino acid sequence): D T VF T T E Y S 5a (general): GA, GA(T/A), (T/C)TC, IGT, IGT, (G/A)AA, IAC,IGT, (G/A)TC. 5b (spec. for bif.): GA, GTA, TTC, (G/A)GT, (G/A)GT, GAA,GAC, (G/A)GT, (G/A)TC. Primer 6 (amino acid sequence): T A M E A V Y 6a(general): TA, IAC, IGC, (T/C)TC, CAT, IGC, IGT. 6b (spec, for bif.):GTA, (G/C)AC, IGC, TTC, CAT, IGC, (G/A)GT.

Example 12

Chromosomal DNA Isolation

Genomic DNA from B.breve 2258 and P.freudenreichii shermani 9093 (PFS)was isolated from 1.5 ml of an overnight broth culture using amodification of the method of Hoffman and Winston (1987). The cells werelysed with glass beads using the procedure as described by Coakley etal. (1996). The DNA pellet was dried at 37° C. in a heating block,resuspended in 50 l sterile destined water and stored at −20° C.Aliquots of 2 μl of extracted DNA were subsequently used in 50 μl PCRreactions.

Example 13

PCR Analysis

PCR amplifications were performed in a total volume of 50 l in a HybaidPCR Express Unit (Hybaid Ltd. Middlesex, UK), with an annealingtemperature of 45° C. Each reaction contained 1 μl of each primer (50pmol/l), 2 μl of template in 5 μl MgCl₂ (50 mM), 5 μl dNTP Master Mix(12.5 mM), 5 μl 10×NH₄ Reaction Buffer and 0.5 μl Biotaq DNA Polymerase(5 u/l) (BIOLINE, London, UK). The resulting amplified 1 kb DNA fragmentwas then cloned into a vector, as described in example 17 (FIG. 4).

Example 14

Chromosome Walking by Inverse PCR

Having confirmed, by comparison of the sequence of the PCR fragment tothe known sequence of linoleic acid isomerase of Lb. reuteri, that theflanking 5′ and 3′ ends of the putative linoleic acid isomerase weremissing the following strategy was followed. The strategy to obtainflanking chromosomal sequence involved the use of two primers, designedto terminal regions of the known chromosomal DNA sequence (FIG. 3). Thegenomic DNA from B.breve 2258 and P. freudenreichii shermanii 9093 weredigested with different restriction enzymes followed by ligation withDNA ligase (1 μl in 50 μl reactions, 400 u/ml, New England BioLabs Inc.Hertfordshire, UK). This was then subsequently used as a template in theinverse PCR reactions with the terminal primers. The reactions were setup the same way as the standard PCR reactions but with an annealingtemperature of 50° C. and the resulting fragment (analysed afterseparation by agarose gel electrophoresis, described in example 16) wascloned into the PCR2.1-TOPO, vector, as described in example 17 (FIG.4). The two terminal primers used were:

Primer A (upstream): 3′ CGTTCTCGACCTTGGTGTTGTATCGGAATT 5′. Primer B(downstream): 5′ GTACCGACCGACAAGATCGAGTCGCTTGCC 3′.

Example 15

Chromosome Walking Using a Single Primer

From the approach described above, it was confirmed that the 3′ end ofthe gene was sequenced, but however, the 5′ end was not obtained. Asecond approach for obtaining the 5′ end of the gene, PCR walkinginvolved the use of just a single primer designed to the 5′ end of thesequenced chrosomal DNA. It was hoped that the primer would bind to this5′ side upstream of the gene and to various sites (at low annealingtemp.), which would generate a number of fragments after PCRamplification. PCR reactions were carried out in a gradient PCR(Stratagene RoboCycler Gradient 96), at 37–50° C. annealingtemperatures. Two reactions (40° C. and 50° C. annealing temp.), eachgenerating a few fragments as evidenced by a small number of bands on anagarose gel (described in example 16) were chosen for cloning, asdescribed in example 17.

Example 16

Analysis of PCR Products by Agarose Gel Electrophoresis

Two microlitres of loading dye was added to 10 μl of each PCR productand loaded on a 1.5% (w/v) agarose (Sigma Chemical CO. St. Louis, Mo.,USA) gel. This DNA was then separated by gel electrophoresis at 100 Vfor 2 h. Gels were stained with ethidium bromide (200 ng/ml in 1× TAEbuffer) and PCR products were visualized by UV transillumination. A 100bp and a 1 kb ladder (New England BioLabs Inc. Hertfordshire, UK) wereused as molecular weight markers.

Example 17

Cloning

Selected PCR fragments based on the genomes of both B.breve 2258 andP.freudenreichii shermanii 9093, were cloned into the vector(pCR2.1-TOPO, Invitrogen BV, Groningen, The Netherlands) which wastransformed, by heat shocking, into competent E.coli cells according tothe manufacturers' instructions (Invitrogen BV, Groningen, TheNetherlands). Recombinants (white colonies) were selected on LB agarsupplemented with 40 μl 5-bromo-4-chloro-3-indolyl-D-galacto-sidase(X-Gal; 40 mg/ml). DNA extraction was performed using QIAGEN PlasmidMini Kit (QIAGEN, Inc., Chatsworth, Calif., USA). Confirmation that thecloning was successful was achieved by digestion with the restrictionenzyme, Eco R1, which has restriction sites within the multiple cloningsite. (FIG. 3 and FIG. 4).

Example 18

Assay other Bifidobacterial Strains for 1 kb PCR Fragment

After obtaining sequencing results of the 1 kb PCR fragment from B.breve2258 and P.freudenreichii shermanii 9093, which confirmed that bothfragments exhibited significant similarity to the linoleic acidisomerase gene sequence of Lb. reuteri, the genomic DNA from a range ofstrains of bifidobacteria (previously isolated as described in example11) was screened (Table 2) for the presence of a similar gene usingprimers 3 (general) and 5 (general) in PCR reactions performed aspreviously described (example 13). The following strains were screened:

-   -   Table 2 Strains screened for the presence of the gene encoding        linoleic acid isomerase using the primers 5 (general) and 3        (general) designed based on the sequence of the gene linoleic        acid isomerase from Lb. reuteri.

Growth in CLA Species Strain Source 0.5 mg/ml prod. B. adolescentis NCFB2204 Adult intestine + 0 B. breve NCFB 2257 Infant intestine + ++ B.breve NCFB 2258 Infant intestine + +++ B. breve NCIMB 8815 Nurslingstools + +++ B. dentium NCFB 2243 Dental carries + +++ B. infantis NCFB2205 Infant intestine + 0 B. lactis Bb 12 Chr. Hansens + 0 B. longumNCFB 2259 Adult intestine + 0 B. longum BB 536 Visby + 0 +++: >60 μgCLA/ml broth ++: >15 μg CLA/ml broth +: >5 μg CLA/ml broth, growth : noCLA produced, no growthThe PCR products obtained following PCR reactions were analyzed byagarose gel electrophoresis, as previously described (example 16).Results and Discussion1. CLA Production by Bacterial Strains

Throughout the screening programme, the two Propionibacterium strains,Propionibacterium freudenreichii subsp. freudenreichii Propioni 6(PFF-6) and Propionibacterium freudenreichii spp. shermanii 9093 (PFS),previously reported to synthesise CLA from linoleic acid (Jiang et al.,1998) were used as positive controls. The CLA biosynthetic assay was setup, with the positive controls in SLM broth, using similar incubationconditions as described previously (Jiang et al., 1998). GLC analysisconfirmed that the two strains did convert free linoleic acid to thecis-9, trans-11 CLA isomer following incubation at 20° C. for 72 h,using CRM (certified reference material) 164 and CIA standards for fattyacid identification (data not shown). However, the levels of CLAproduced by the two strains of Propionibacterium were lower than thatreported previously by Jiang et al. (1998), producing ˜60 μg/ml of CLAin comparison with 111.8 μg/ml previously reported by Jiang et al.(1998), using 0.5 mg/ml linoleic acid as substrate. In addition, wefound that the amount of linoleic acid remaining in the media followingincubation with the PFS strain was ˜50 μg/ml, compared with 289.5 μg/mlreported previously (Jiang et al., 1998). The variation in these datamay be a result of differences in the numbers of cells present duringincubation, and possibly as a result of the different procedures usedfor fatty acid extraction and methylation.

Three strains of Propionibacterium were then examined for their abilityto produce CLA. These were Propionibacterium acidi propionici NCFB 5633,Propionibacterium freudenreichii spp. shermanii LMG 16424 andPropionibacterium freudenreichii spp. shermanii JS (Laboratorium Visby,Tonder, Denmark). The strains were incubated in the presence of 0.5mg/ml linoleic acid using the same growth conditions and media asdescribed above. The two Propionibacterium shermanii strains synthesizedCLA in MRS media while Propionibacterium acidi propionici did notproduce any detectable CLA product (Table 1). The amounts of CLAproduced by the two Propionibacterium shermanii strains (12–14 μg/ml)were low however, compared with 60 μg/ml produced by PFS strain in thisstudy.

2. Screening of Lactobacilli, Lactococci, and Pediococci for CLAProduction

A variety of different strains of lactobacilli, lactococci, andpediococci, obtained from various sources (Table 3) were tested forability to produce CLA from linoleic acid. These strains included anumber of probiotic strains including five strains of Lb.reuteri and thebacteriocin producing Lactcoccus lactis DPC 3147 strain (Ryan et al.,1996). The strains were inoculated into MRS to a density of ˜10⁶ cfu/mland incubated under respective conditions as described above, in thepresence of linoleic acid concentrations of 0.5 to 3.0 mg/ml.

The ability of the strains to grow in the different linoleic acidconcentrations varied considerably. Good growth of all five Lb.reuteriistrains occurred at linoleic acid concentrations up to 1 mg/ml, while at3 mg/ml, the growth of strains NCIMB 701359 and NCIMB 70256 wascompletely inhibited (data not shown). At all linoleic acidconcentrations investigated, none of the Lb.reuteri strains investigatedproduced CLA in detectable quantities. Lactobacillus helveticus NCDO1244 exhibited no growth in 0.5 mg/ml linoleic acid while Lactobacillusleichmanii NCDO 302 showed good growth in the presence of high linoleicacid concetrations (3 mg/ml). However, none of the strains produced CLAfrom linoleic acid (between 0.5 to 3 mg/ml) under the conditions used.

TABLE 3 Strains screened for CLA production Species Code SourceLactobacillus reuteri NCIMB 11951 Adult intestine Lactobacillus reuteriNCIMB 701359 Unknown Lactobacillus reuteri NCIMB 701089 UnknownLactobacillus reuteri NCIMB 702655 From rat Lactobacillus reuteri NCIMB702656 From rat Lactobacillus reuteri DSM 20016 Lactobacillus helveticusNCDO 257 Lactobacillus helveticus ATCC 15009 Lactobacillus helveticusNCDO 1244 Lactobacillus leichmanii NCDO 299 Lactobacillus leichmaniiNCDO 302 Lactobacillus fermenticum ATCC 338 Lactobacillus acidophilusATCC 4356 Lactobacillus DPC 5336 from cracker Barrel Bifidobacteriumbreve NCTC 11815 Lactcoccus lactis DPC 3147 Lactococcus lactis 290P DPC152 Pediococcus pentasescus FBB 633. CLA Production from Linoleic Acid Among Bifidobacterium StrainsCultured in MRS

A variety of bifidobacteria obtained from a number of sources (Table 4)were screened for CLA production. Since free linoleic acid was found tobe inhibitory to the growth of bifidobacteria strains, the minimuminhibitory concentration of linoleic acid for the B.breve strains wasinitially determined. This involved inoculation (1% from grown cultures)of the Bifidobacterium strains into MRS containing free linoleic acidconcentrations ranging from 0.2 to 1.5 mg/ml and incubation underanaerobic conditions at 37° C. for 48 h. The pH of the media remainedunchanged following the addition of the linoleic acid substrate in thisconcentration range at pH˜6.1. Viable bifidobacteria were enumerated attime zero and following 48 h incubation in the presence of the linoleicacid substrate. Viability of B.breve 2257 was unaffecetd in the presenceof linoleic acid, at concentrations up to 0.5 mg/ml. However, viabilitywas dramatically reduced at 1.0 mg/ml and only 1.5% survival wasobserved. In contrast the survival of strains B.breve 2258, 8807, 8815and 11815 was reduced to <60% at linoleic acid concentrations of 0.2mg/ml and higher. The bifidobacteria strains were then screened for CLAproduction from linoleic acid substrate at a concentration of 0.5 mg/ml,using the incubation conditions described above. A number of theBifidobacterium strains investigated produced CLA following incubationin MRS containing 0.5 mg/ml linoleic acid, and the results from thisscreening program showed that there was considerable interspeciesvariation in the ability of bifidobacteria to produce CLA (Table 4).

TABLE 4 Conversion to CLA by Bifidobacterium strains cultured in MRSbroth containing cysteine spiked with 0.5 mg/ml linoleic acid for 48 h.Growth in 0.5 Species prod. Strain Source mg/ml CLA B. aolescentis NCFB2204 Adult intestine +¹ 0 B. adolescentis NCFB 2230 Adult intestine 0² −³ B. adolescentis NCFB 2231 Adult intestine +  0⁴ B. angulatum NCFB2236 Human faeces + 0 B. bifidum NCFB 795 Human milk + 0 B. breve NCFB2257 Infant intestine + ++ B. breve NCFB 2258 Infant intestine + +++⁷ B.breve NCTC 11815 Infant intestine + +++ B. breve NCIMB 8815 Nursingstools + +++ B. breve NCIMB 8807 Nursing stools + +++ B. dentium NCFB2243 Dental carries + +++ B. infantis NCFB 2205 Infant intestine + 0 B.infantis NCFB 2256 Infant intestine + 0 B. lactis Bb12 Chr. Hansens + 0B. longum BB536 Visby + 0 B. pseudocatenulatum NCIMB 8811 Nurslingstools + + ¹growth ²no growth ³not determined ⁴no CLA produced 5. >5μg/ml CLA 6. >15 μg/ml CLA ⁷>60 μg/ml of brothAll 5 strains of Bifidobacterium breve species examined tested positivefor CLA production with four of these strains producing more than 60μg/ml CLA, while strain B.breve NCFB 2257 produced 15 μg/ml under theseconditions. In addition, B.dentium NCFB 2243 was an efficient CLAproducer, also yielding >60 μg/mg CLA (Table 4), whileB.pseudocatenulatum NCIMB 8811 produced >15 μg/ml under the experimentalconditions employed. Among the other bifidobacteria speciesinvestigated, 3 strains of B.adolescentis, 2 strains of B.longum and 1strain each of B.angulatum, B.bifidum and B.lactis were all negative forCLA production (Table 4). The exact role of biohydrogenation in themetabolic environment of the bacterial cell is unclear. In the study byJiang et al. (1998), strains which were able to produce CLA were thoseinhibited by the presence of free linoleic acid, but a positivecorrelation between CLA production and tolerance to linoleic acid wasobserved within the three CLA producing strains of propionibacteria.This suggests that the conversion of linoleic acid to CLA is adetoxification mechanism for the bacterial cell. This is supported bythe fact that the anti-microbial activity of fatty acids with doublebonds of cis configuration is stronger than that of trans (Kabara,1983).

The most efficient CLA producers were strains B. breve 8815 and 2258 atlinoleic acid concentration of 1.0 mg/ml. The strains B. breve 8815,2258 and 2257 were present at less than 10⁴ cells/ml at the highestlinoleic acid concentration (1.5 mg/ml) and were not analysed for CLAconversion. B.breve NCFB 2258 converted ˜50% of the added linoleic acidto CLA at 0.2 and 0.5 mg/ml linoleic acid concentrations. The ability ofB.breve NCFB 2258 strain to utilise triglyceride bound linoleic acid assubstrate for CLA production, using trilinolein. (C_(18:2), cis-9,cis-12), sunflower and soybean oils was also investigated. The B.breveNCFB 2258 strain was found to be negative for ability to utilisetriglyceride bound linoleic acid as substrate at 0.2 mg/ml linoleic acidfor CLA production (data not shown), from trilinolein, sunflower andsoybean oils. These data are in agreement with a previous study whichshowed that of 61 rumen isolates with ability to produce CLA, noneutilised triglyceride bound linoleic acid (Fujimoto et al., 1993). Inaddition, trilinolein did not inhibit the growth of B.breve 2258 to thesame extent as similar concentrations of free linoleic acid (data notshown). This indicates that linoleic acid in the free fatty acid form ismore toxic to bifidobacteria than triglyceride-bound linoleic acid.

4. Microbial Biohydrogenation of Unsaturated C 18 Fatty Acids

B.breve 2258 and P.freudenreichii shermanii 9093 were screened for theirability to produce CLA. The strains were incubated in duplicate in thepresence of 0.5 mg/ml linoleic acid (LA) and CLA (the pure cis-9,trans-11 isomer), respectively, using the same growth conditions andmedia as described. In order to compare the fatty acid composition,control cultures without added LA and CLA were also incubated using thesame conditions. The propionibacteria strain used in this study waspreviously reported to synthezise CLA from LA (Jiang et al., 1998) andtherefore used as a positive control. However, in this study, thatresult was not reproducible since the strain was clearly inhibited inthe presence of LA and CLA and hence grew very poorly. No CLA productionfrom that strain was therefore detected and the results from the GLCanalysis are not presented here.

After separation (by centrifugation) of B.breve 2258 cells from thesupernatant following incubation for 48 h, followed by lipid extractionand methylation, the fatty acid composition of both the cells (pellets)and the supernatant were analysed using GLC (example 10).

5. Change in Supernatant Fatty Acid Composition Following Incubation ofB.breve 2258 with 0.5 mg/ml LA

GLC analysis confirmed that B.breve 2258 converted LA to CLA. Of theadded 0.5 mg/ml, only 0.27 mg/ml (54%) remained in the supernatant (FIG.5), while the remainder (46%) was converted to other fatty acids,preferentially the cis-9, trans-11 CLA isomer followed by cis-9-C 18:1(oleic acid) and a peak identified as trans-9, trans-11-CLA. The amountof cis-9, trans-11 CLA produced was 0.136 mg/ml, and trans-9,trans-11-CLA accounted for 0.03 mg/ml. The amount of these two fattyacids present in the control supernatant was negligible (FIG. 5) Therewas also a substantial increase of cis-9-C 18:1 (oleic acid) (64.8%compared with the control supernatant), which indicates that B.breve2258 harbours a CLA reductase enzyme that hydrogenates the trans-11double bond of cis-9, trans-11 CLA. Compared to the control culturethere was64,8% more stearic acid in the LA added supernatant. Smallerincreases were observed also in the concentrations of trans-11 C 18:1(vaccenic acid) (30.3%) and C 18:0 (stearic acid) (17.5%) compared withthe control supernatant, suggesting that other hydrogenating enzymes maybe involved.

6. Change in Membrane Fatty Acid Composition Following Incubation of B.breve 2258 with 0.5 mg/ml LA

The fatty acid composition of the membranes from the cultures (pellet)grown in MRS medium with 0.5 mg/ml LA was also analysed and comparedwith the control cultures (FIG. 9). Results are expressed as mg fattyacids from cells/ml fully grown culture. The fatty acid concentration inthe pellets in mg/ml is lower than that of the supernatant and thereforeare not directly comparable. Results from the GLC analysis show that CLAwas incorporated in the cell membranes, whereas the control culturecontained negligible CLA. The cis-9, trans-11 isomer was the mostabundant CLA isomer and accounted for 0.012 mg/ml, which represents 70%of the total CLA isomers. The content of the cis-9-C 18:1 (oleic acid)was increased (by 271% compared with controls) in the membranes ofB.breve 2258 cells incubated in LA (0.5 mg/ml), indicating the presenceof a CLA reductase, which was capable of reducing the unsaturatedtrans-11 bond in CLA in B.breve 2258. The trans-11 C 18:1 (vaccenicacid) content of the cell membranes wes reduced (over 4-fold) in the LAtreated cells compared with the control cells. As seen in thesupernatant, a small increase of 28% in C 18:0 (stearic acid) wasdetected in the membranes of the LA treated cells compared with thecontrols (FIG. 9).

7. Change in Supernatant Fatty Acid Composition Following Incubation ofB.breve 2258 with 0.5 mg/ml cis-9, trans-11 CLA

In order to evaluate if B.breve 2258 possesses enzymes other than theputative linoleic acid isomerase, involved in the biohydrogenation oflinoleic acid, studies were undertaken using cis-9, trans-11 CLA as thesubstrate. Strain B.breve 2258 was inoculated in MRS containing 0.5mg/ml of the pure cis-9, trans-11 CLA isomer (Matreya Inc. PA, USA) andincubated for 24 h at 37° C. Following incubation in the presence CLA(0.5 mg/ml), only 0.32 mg/ml (65%) remained in the supernatant (FIG. 9)with the remaining 35% converted to other fatty acids. The mostpredominantly formed fatty acid corresponded to trans-9, trans-11-CLA,observed following incubation with LA and eluted at 43 mins (FIGS. 6 and10). This CLA isomer was present at 0.12 mg/ml (71% of the cis-9,trans-11 CLA peak). Oleic acid was also formed in the supernatant byB.breve 2258 following incubation with cis-9, trans-11 CLA with anincrease of 85.5% compared with the control supernatant. Smallerincreases were also observed in the concentration of trans-11-C 18:1(vaccenic acid) (74.5% compared to control.supernatant) and C 18:0(stearic acid) (23.9% compared to control supernatant).

8. Change in Membrane Fatty Acid Composition Following Incubation ofB.breve 2258 with 0.5 mg/ml cis-9, trans-11 CLA

The lipid composition of the membrane following incubation of B.breve2258 in cis-9, trans-11 CLA was also compared with control cellsincubated in the absence of CLA. The fatty acid composition of themembranes from cultures inoculated in MRS containing 0.5 mg/ml of thepure cis-9, trans-11 CLA isomer shows that the membrane compositionchanged compared with the control. Cis-9, trans-11 CLA was incorporatedinto the membrane of the culture grown in the presence of CLA (0.03mg/ml) compared with the culture, grown in the absence of CLA, whichcontained no CLA (FIG. 12.). The trans-9, trans-11-CLA as observed inthe supernatant was also present in the membrane following incubationwith cis-9, trans-11 CLA (0.012 mg/ml). Clearly the bacterial cell hasthe capacity to convert cis-9, trans-11 CLA to trans-9, trans-11-CLA. Anincrease of 54.8% in the cis-9-C 18:1 oleic acid membrane content wasobtained following incubation of B.breve 2258 in CLA (0.5 mg/ml). Thisamount of oleic acid in the membranes, formed relative to the control,was greater in the LA treated cells (2.7-fold increase) than the CLAtreated cells. As observed in the cell membranes obtained followingincubation of B.breve 2258 in LA (0.5 mg/ml), the trans-11-C 18:1vaccenic acid content of membranes was lower in the cells incubated withcis-9, trans-11 CLA than the control pellets (5.8-fold greater in thecontrol). Only a very small increase was obtained in the content of C18:0 stearic acid in the membranes of CLA treated cells compared withcontrols.

The GLC analysis confirmed that B.breve 2258 converted LA to CLA andthat a significant amount trans-9, trans-11-CLA was also formed and thedata also indicates that a further biohydrogenation of cis-9, trans-11CLA to C 18:1 isomers, preferably the cis-9-C 18:1 isomer occurs as aresult of incubation with B.breve 2258 strain. Since also a smallincrease of C 18:0 was detected in the chromatogram, it is possible thatadditional enzymes are involved, but whether this activity issignificant is unclear. The increase in saturation was obtained in boththe supernatant and the bacterial pellets. Also when the pure CLA isomerwas incubated with B. breve 2258 it was further hydrogenated to moresaturated fatty acids, primarily cis-9-C 18:1. This may support thetheory that incorporation of a trans fatty acids instead of cis, andsaturation or trans conversion, of cis double bonds is a strategy forthe bacterial cell to counteract for the increased fluidity that occurswhen LA and the cis-9, trans-11 CLA isomer (which has a cis bond) isinterfering with the membranes which leads to expansion of membrane,elevation of membrane permeability and impairment of membrane functions(Junkers and Ramos, 1999; Weber et al., 1994).

Interestingly in this study, the differences in fatty acid compositionwhen adding LA and CLA respectively, to the supernatant and pellets, isnot very significant. When adding the pure cis-9, trans-11 CLA isomer tothe supernatant it is converted to a great extent to other CLA isomer,which is not the case in the LA added supernatant.

Because of all the beneficial health effects of CLA, the ability ofstrains of bifidobacteria, natural inhabitants of the intestine, toconvert free linoleic acid to CLA can be considered as a novel probiotictrait. Indeed, it is tempting to suggest that the anticarcinogenicactivity ascribed to some of these probiotic bacteria could be linked totheir ability to produce CLA. Development of probiotic dairy productswith elevated CLA levels also provides an exciting opportunity.Exploitation of probiotic bifidobacteria harbouring CLA biosyntheticcapabilities offers novel opportunities in the rational design ofimproved health-promoting functional foods, with the benefits ofenriched CLA and probiotic bacteria.

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1. An isolated nucleic acid molecule which encodes a polypeptide withconjugated linoleic acid isomerase activity, or the full complementthereof, selected from the group consisting of: a) a nucleic acidmolecule comprising the nucleotide sequence set forth in SEQ ID NO: 1,b) a nucleic acid molecule comprising a nucleotide sequence whichencodes a polypeptide sequence comprising the amino acid sequence as setforth in SEQ ID NO:2, c) a nucleic acid molecule comprising a nucleotidesequence which is a derivative of the nucleic acid sequence shown in SEQID NO: 1, which encodes a polypeptide having at least 98% identity tothe entire amino acid sequence as set forth in SEQ ID NO:2 withoutsubstantially reducing the enzymatic activity of the polypeptide; and d)a nucleic acid molecule comprising a nucleotide sequence which is atleast 95% identical to the entire nucleotide sequence set forth in SEQID NO:1 and encodes a polypeptide having conjugated linoleic acidisomerase activity.
 2. A nucleic acid construct comprising a nucleicacid molecule of claim 1, where the nucleic acid molecule is linked toone or more regulatory signals.
 3. A vector comprising a nucleic acidmolecule of claim 1 or the nucleic acid construct of claim
 2. 4. Atransgenic microorganism comprising at least one nucleic acid moleculeof claim 1 or at least one nucleic acid construct of claim
 2. 5. Thetransgenic microorganism of claim 4, which is a bacterium.
 6. A processfor the production of conjugated linoleic acid comprising cultivating arecombinant microorganism into which the nucleic acid molecule of claim1 or the nucleic acid construct of claim 4 has been introduced in thepresence of linoleic acid and isolating the formed conjugated linoleicacid.
 7. The process of claim 6 wherein the conjugated linoleic acid iscis-9, trans-11 octadecadienoic acid.
 8. The process of claim 6 whereinthe microorganism is selected from the group consisting ofBifidobacterium breve, Bifidobacterium dentium and Bifidobacteriumpseudocatenulatum.
 9. The process of claim 6 wherein the concentrationof conjugated linoleic acid in the culture medium is higher than 1mg/ml.
 10. A process for the production of conjugated linoleic acidwhich comprises introducing at least one nucleic acid sequence asclaimed in claim 1 or at least one nucleic acid construct as claimed inclaim 2 into an oil-producing organism, growing this organism, isolatingthe oil contained in the organism and liberating the fatty acidscontained in the oil.
 11. A process for the production of triglycerideswith an increased content of conjugated linoleic acid, which comprisesintroducing at least one nucleic acid sequence as claimed in claim 1 orat least one nucleic acid construct as claimed in claim 4 into anoil-producing organism, growing this organism and isolating the oilcontained in the organism.
 12. The process of claim 10, wherein theorganism is a microorganism.
 13. The process of claim 10, wherein theorganism is a microorganism of the genera Bifidobacterium,Propionibacterium, Lactobacillus or Butyrivibrio.
 14. The process ofclaim 10, wherein the organism is a microorganism of the genusBifidobacterium.
 15. The process of claim 6, wherein the microorganismis of the genus Bifidobacterium.
 16. The process of claim 7 wherein themicroorganism is selected from the group consisting of Bifidobacteriumbreve, Bifidobacterium dentium and Bifidobacterium pseudocatenulatum.17. The process of claim 7 wherein the concentration of conjugatedlinoleic acid in the culture medium is higher than 1 mg/ml.